This proposal aims to discover the role of an E3 ubiquitin ligase, EDD, in mediating signaling downstream of RIP1 in a novel necrotic cell death pathway, termed necroptosis. Necroptosis is activated by the autocrine release of TNFa through RIP1 kinase dependent mechanism. An increased release of TNFa has been shown to occur in multiple age-related chronic neurodegenerative diseases such as Alzheimer's disease. Cells from older individuals have an increased propensity to undergo necrosis, instead of apoptosis, in response to stress. The hypothesis is that neuronal cell death in chronic neurodegenerative diseases, such as Alzheimer's disease, is primarily necrosis, rather than apoptosis. Necroptosis is the only cellular mechanism identified to mediate necrotic cell death, EDD was identified in an siRNA screen as a mediator of necroptosis. EDD interacts with R1P1 and can be phosphorylated by RIPI. The Specific Aim 1 is to characterize EDD as the first substrate of RIPI in mediating necroptosis. The phosphorylation site on EDD by RIPI will be identified and used to generate a phospho-EDD antibody. This antibody will be used to study the dynamics of EDD phosphorylation during necroptosis and to probe mouse brain lysates from APP23 transgenic mice to determine if RIPI is activated in Alzheimer's disease. The Specific Aim 2 is to determine if the ubiquitination activity of EDD negatively regulates RIPI as a negative feedback control. The interacting domains of EDD and RIPI will be identified as well as the dynamics and localization of the interaction. The Specific Aim 3 is to characterize the role of EDD in mediating TNFa release. The temporal impact of EDD knockdown on TNFa release will be studied by ELISA. EDD truncation mutants will be used to determine if its PABC domain, which interacts the cellular protein translation machinery, is involved in mediating TNFa release by reconstituting the truncated EDD in EDD knockdown cells. A general role of EDD in mediating TNFa release in Alzheimer's disease will be examined by checking if EDD knockdown in primary microglia blocks TNFa release induced by |3-amyloid peptide. The studies outlined above may reveal EDD as a new target for treatment of neuroinflammation involved in chronic neurodegenerative diseases such as Alzheimer's disease. Necrosis is involved in age-related neurodegeneration, such as Alzheimer's disease. This proposal will study the mechanism of a novel necrotic cell death pathway and its importance in neurodegenerative disease.